3D directional tuning in the orofacial sensorimotor cortex during natural feeding and drinking

Directional tongue movements are essential for vital behaviors, such as feeding and speech, to position food for chewing and swallowing safely and to position the tongue for accurate sound production. While directional tuning has been well-studied in the arm region of the sensorimotor cortex during reaching tasks, little is known about how 3D tongue direction is encoded in the orofacial region during natural behaviors. Understanding how tongue direction is represented in the brain has important implications for improving rehabilitation for people with orolingual dysfunctions. The goal of this study is to investigate how 3D direction of tongue movement is encoded in the orofacial sensorimotor cortex (OSMCx) during feeding and drinking, and how this process is affected by the loss of oral sensation. Using biplanar video-radiography to track implanted markers in the tongue of behaving non-human primates (Macaca mulatta), 3D positional data was recorded simultaneously with spiking activity in primary motor (MIo) and somatosensory (SIo) areas of the orofacial cortex using chronically implanted microelectrode arrays. In some sessions, tasks were preceded by bilateral nerve block injections to the sensory branches of the trigeminal nerve. Modulation to the 3D tongue direction was found in a majority of MIo but not SIo neurons during feeding, while the majority of neurons in both areas were modulated to the direction of tongue protrusion during drinking. Following sensory loss, the proportion of directionally tuned neurons decreased and shifts in the distribution of preferred direction were observed in OSMCx neurons. Overall, we show that 3D directional tuning of MIo and SIo to tongue movements varies with behavioral tasks and availability of sensory information.


Introduc on
Motor and somatosensory cor cal neurons have long been known to modulate their spiking ac vity to the direc on of arm movements during reaching tasks performed by non-human primates (Georgopoulos et al., 1988;Schwartz et al., 1988a;Prud'homme and Kalaska, 1994).Similarly, neurons in the primary motor (MIo) and primary somatosensory (SIo) areas of the orofacial sensorimotor cortex (OSMCx) have been shown to modulate their spiking ac vity to the direc on of voluntary tongue protrusion (Murray and Sessle, 1992;Lin et al., 1994) and to semiautoma c movements, such as chewing and swallowing (Sessle et al., 2005b).However, considerably less is known about how 3D tongue direc on is encoded in the orofacial region during natural feeding and drinking behaviors.Due to the tongue being inside the oral cavity and thus hidden from external view, it has proved difficult to study the neuromechanical processes underlying important behaviors such as feeding, drinking, and speech.This is significant for people with sensorimotor dysfunc ons in chewing, swallowing, or speaking.Because specific direc onal tongue movements are essen al for such behaviors, understanding how tongue direc on is represented in the brain may help improve evalua on and treatment strategies.
The seminal studies on the direc onal tuning proper es of OSMCx neurons by Sessle and colleagues employed varying loca ons of spouts that delivered a juice reward to elicit direc onal tongue protrusions without tracking tongue movements.A later study incorporated tracking of 2D tongue movements using videofluoroscopy during voluntary direc onal protrusions (Arce et al., 2013), but the tongue trajectories were not used to study direc onal tuning.In all these prior studies, primates have been trained to interact with a computer display to elicit a tongue protrusion to a specific direc on on cue.There is a knowledge gap on how spiking ac vity in the OSMCx relates to tongue movements during natural behaviors.With the development of biplanar video-radiography (Brainerd et al., 2010), it is now possible to track these 3D tongue movements within the oral cavity at a high resolu on.By simultaneous recording of tongue movements and spiking ac vity, we have shown recently that tongue posi on and shape can be accurately decoded from OSMCx during feeding (Laurence-Chasen et al., 2023).
In this study, we examined both the encoding and decoding of tongue direc on in the OSMCx during untrained feeding and drinking and how this differed across mul ple cor cal regions.Because oral somatosensa on is vital for proper tongue posi oning and bolus control, and when it is impaired can cause difficulty in chewing and swallowing (Smith and Cutrer, 2011), we also inves gated the role of tac le feedback in the sensorimotor control of the tongue during these behaviors.

Methods
Experimental setup.Experiments were performed on two adult male rhesus macaques (Macaca mula a, 9-10 kg, ages 8 and 9 years) in the University of Chicago XROMM Facility.This sample size was chosen based on precedent in the field of non-human primate motor neuroscience.All protocols were approved by the University of Chicago Animal Care and Use Commi ee and complied with the Na onal Ins tutes of Health Guide for the Care and Use of Laboratory Animals.The subjects were seated in a standard primate chair and head-fixed to keep their head posi on constant during feeding and drinking trials.Each trial lasted 10 seconds.In a feeding trial, a piece of food (grape, gummy bear, pasta) of roughly the same size was presented directly to the animals' mouth using a stylus.In a drinking trial, juice was delivered through one of three spouts posi oned in front of the subject (Fig. 1A).
For some sessions, these behavioral tasks were preceded by nerve block injec ons (0.25% Bupivacaine HCL and Epinephrine 1:200,000, 0.25 mL/injec on site) to the sensory branches of bilateral trigeminal nerves (lingual, inferior alveolar, buccal, pala ne) to eliminate oral tac le sensa on locally and temporarily.The nerve block was administered while the subjects were under general anesthesia, and all data were collected within 90 minutes of the nerve block.Each monkey served as its own control, with nerve block feeding data collec on sessions taking place either a day before or a day a er the associated control session.Nerve block drinking data collec on was performed immediately following the control drinking session.Mul ple datasets (40-60 trials) were collected for both subjects across mul ple days.However, due to the complex and me-consuming nature of processing integrated XROMM and neural data, one session per subject, behavior, and condi on was used for this study.Thus, we analyzed a total of 8 datasets.

Video-radiography.
Prior to data collec on, the animals were implanted with spherical tantalum beads (1-mm diameter) in the cranium, mandible, and the tongue, from the p to the region of the circumvallate papillae.During feeding or drinking, the movement of these markers was recorded using high-resolu on (200 Hz, <0.1 mm) biplanar video-radiography collected with Xcitex ProCapture version 1.0.3.6.The 3D posi onal data was obtained following the previously described X-ray Reconstruc on of Moving Morphology (XROMM) workflow (Laurence-Chasen et al., 2020) incorpora ng the use of XMALab (Knörlein et al., 2016) and machine learning using DeepLabCut (Mathis et al., 2018) to reconstruct the kinema c data.The , , values of the markers were then smoothed with a 30 Hz low-pass Bu erworth filter and transformed into a cranial coordinate space with the origin fixed at the posterior nasal spine.Gape cycles within each feeding sequence were manually iden fied and categorized by cycle type (manipula on, stage 1 transport, chew, stage 2 transport, or swallow).
Electrophysiology.Under general anesthesia, a microelectrode array was chronically implanted in four areas of the le hemisphere (Supplementary file 1, Fig. 1): rostral MIo (96-electrode Utah array; electrode length: 1.5 mm), caudal MIo (32-electrode Floa ng microelectrode array (FMA), electrode length: 3.0-4.5 mm), area 1/2 (96-electrode Utah array, electrode length: 1.0 mm), and area 3a/3b (32-electrode FMA, electrode length: 4.0-8.7 mm).The neural data was recorded using Grapevine Neural Interface Processor (Ripple Neuro, Salt Lake City, UT).Signals were amplified and bandpass filtered between 0.1 Hz and 7.5 kHz and recorded digitally (16-bit) at 30 kHz per channel.Only waveforms (1.7 ms in dura on; 48 sample me points per waveform) that crossed a threshold were stored and offline spike sorted (Offline Sorter, Plexon, Dallas, TX) to remove noise and to isolate individual neurons.The channel name assigned to each recorded neuron was kept consistent between control and nerve block data for comparison.Data analysis.3D tongue kinema cs were recorded simultaneously with the neural data in all behavioral sessions.All data analyses were performed in MATLAB 2022b (MathWorks, Na ck, MA).For feeding, the instantaneous 3D direc on of the tongue p marker for every 100 ms throughout each gape cycle was calculated as: Where is the , , posi on at the start of each 100-ms interval and is the posi on at the end (Fig. 1B).These direc ons were then categorized based on whether the movement was nega ve or posi ve rela ve to the horizontal plane (Le /Right), the sagi al plane (Inferior/Superior), and the axis (Posterior/Anterior).This resulted in eight direc ons: AntSupL, AntSupR, AntInfL, AntInfR, PostSupL, PostSupR, PostInfL, and PostInfR.An equal number of 100ms intervals from each of these direc ons was sampled, and spike data during each was used for neural analysis.For comparison with the drinking task, the sign was determined rela ve to the horizontal plane, with rightward tongue movement being posi ve.This is also the plane of mo on which has been the least studied.These le -right direc ons were categorized into six 10-bins with a total range of -30 to 30, which encapsulated the majority of the observed distribu on of direc ons in each subject.Lingual yaw (transverse rota on) and pitch (eleva on/depression) were also calculated to compare tuning across the lateral and ver cal components of tongue direc on (Supplementary file 2, Equa on 2).For drinking, the direc on was determined by which of the three spouts juice was dispensed from during each lick.The spiking ac vity used for neural analysis of the drinking task was from intervals of ±250 ms around each minimum protrusion of the tongue.As 100 ms was not sufficient to capture the full range of tongue mo on during each drinking cycle, the length of me used was increased to allow a clear dis nc on between the three direc ons.Kinema c performance for feeding was determined by the spread of tongue direc ons observed across trials.For drinking trials, performance was determined by the variance of endpoint posi ons as well as by the propor on of "failed" cycles, where the monkey missed the correct spout loca on with their tongue p.The difference between control and nerve block performance was evaluated using a two-tailed t-test and f-test.
Tongue direc ons were subsequently compared with the firing rates of individual neurons across cor cal areas.Neurons were iden fied as direc onally modulated if their mean firing rate varied significantly across direc ons (Kruskal-Wallis, p < 0.05).The propor ons of neurons that were found to be direc onally tuned were compared across groups using a chi-square test.Then, mul ple linear regression was used to determine if the firing of each neuron fit the cosine tuning func on that has been previously described for the arm area of the motor cortex (Schwartz et al., 1988b).To accomplish this the direc onal components of a unit vector represen ng each group of direc ons were calculated.For neurons that fit the tuning func on, a preferred direc on (PD) in 3D space was es mated.These PDs are distributed around a unit sphere, with the origin represen ng the start of the movement.The direc onal index was calculated as a measure of the depth of direc onal tuning.The PD for the drinking task was determined as the direc on for which a neuron exhibited its maximal firing rate.Similarly, a PD across the le -right feeding direc ons was determined for comparison.Circular concentra on (k-test) to compare distribu ons of PDs during feeding and polar plot genera on (Fig. 6A and Fig. 10A) were performed using the CircStat MATLAB toolbox (Berens, 2009).For drinking, distribu ons of PDs were compared using a chi-square test.
Decoding tongue direc on.The ability to predict tongue direc on from spiking ac vity of MIo and SIo neurons was evaluated using a K-nearest neighbor (KNN) classifier.The Euclidean distance was used to iden fy nearest neighbors, and the number of nearest neighbors used was K = 7.This K value was determined a er tes ng different Ks which yielded comparable results.The feature was the firing rate of each neuron over each trial: every 100 ms throughout feeding sequence, or 100 ms centered at minimum tongue protrusion during drinking.As a more direct comparison to the drinking, feeding direc ons were combined into three groups represen ng le , middle, and right movement direc ons.The decoder was trained on 80% of trials and tested on the remaining 20%, then decoder performance was determined by the percentage of test trials where the direc on of movement was correctly decoded from the neural data.We ran 100 itera ons of the classifier using a different set of randomly selected training and test trials then calculated the average performance.The same sets of training and test trials were used for decoding from simultaneously recorded MIo and SIo data.However, our recorded popula ons were of variable sizes, and decoding performance was found to be related to the number of neurons in the ensemble (Supplementary file 1, Fig. 6).Because the smallest popula on of neurons we recorded was 28, we selected 28 random neurons from the larger popula ons for each itera on.Based on the posi ve rela onship between popula on size and decoding accuracy, we expect that performance would increase with more neurons.These results will show whether tongue direc on can be decoded from a very small number of neurons.We fit a linear regression model with interac ons to compare decoding performance across the other variables in the experiment.

Results
Past studies have inves gated direc onal tuning of OSMCx neurons during trained tongueprotrusion tasks.Here we study two untrained and natural behaviors, feeding and licking (i.e., "drinking") from a spout.In the feeding task, there is no guidance on how to move the tongue, whereas in the drinking task, the direc on of movement is guided by the loca on of the spout.By comparing how modula on to tongue direc on differs between natural tasks with and without specific direc onal input, we can examine how the movement of the tongue is coordinated by the OSMCx under different behavioral contexts.

Figure 2. Examples of single neuron activity in relation to tongue direction. (A) Each peri-event time histogram (PETH and ±1 SE, smoothed by a 50-ms Gaussian kernel) corresponds to spiking activity during a 200 ms window for a specific range of tongue direction for feeding trials. Dashed lines indicate the 100-ms interval used for calculating the tongue direction. (B) PETHs for drinking trials with the same spout, centered at the point of minimum protrusion of the tongue (0 s).
In both tasks, many neurons exhibited significant modula on to tongue direc on (Kruskal-Wallis, p < 0.05), though there were diverse pa erns of spiking ac vity.Figure 3A illustrates the direc onal tuning for a neuron in MIo during natural feeding.In this example the neuron is strongly tuned to posterior-anterior and inferior-superior movement direc ons but shows no strong preference across the le -right axis.Many of the recorded neurons in each popula on behaved in a similar fashion, with peaks most frequently observed toward the anterior and superior direc ons.In the feeding task, over 80% of neurons in the motor areas (rostral and caudal MIo) exhibited tuning to 3D direc on (Fig. 3B, le ), compared to only over 40% of neurons in the somatosensory areas (areas 3a/3b, 1/2).The difference in the propor on of MIo and SIo neurons exhibi ng direc onal tuning during feeding was significant in both subjects (Chi-square, p < 0.001).Fewer neurons were tuned during swallows than chews (Supplementary file 1, Fig. 4; MIo: p < 0.05, SIo: p > 0.1).In the drinking task, greater than 50% of neurons across all recorded areas in both subjects exhibited tuning to the direc on of tongue protrusion (Fig. 3B, right).Unlike the feeding task, the difference between the propor on of direc onally tuned neurons in MIo and SIo was not significant in the drinking task for either subject (Chi-square, p > 0.1).We then used mul ple linear regression to determine whether the firing of these neurons varied in an orderly fashion with tongue direc on.Of the neurons that were found to be direc onally modulated during feeding, 86.36% in MIo and 75.32% in SIo also fit the tuning func on (F-test, p < 0.05).The distribu on of preferred direc ons was non-uniform across the unit sphere (Fig. 4A; Rayleigh test, p < 0.001), with the highest density of PDs toward the most inferior and superior direc ons.The distribu on of the direc onal index was skewed with a mean of 0.5374 (Fig. 4B).We also analyzed the tuning of these neurons to the lateral (yaw) and ver cal (pitch) components of tongue direc on during feeding.Figure 5A shows peak ac vity of a neuron in MIo and in SIo at varying degrees of pitch and yaw.Overall, there was a higher propor on of neurons tuned to pitch than yaw (Fig. 5B).More neurons in MIo than in SIo exhibited tuning to both yaw and pitch (Chi-square, yaw: p < 0.08, pitch: p < 0.001), consistent with our 3D direc on results.Of those neurons that were direc onally tuned, we generally observed sharper and more narrow tuning curves in MIo, compared to the broader tuning of SIo (Fig. 5A).More informa on about direc onal tuning to yaw and pitch is included in Supplementary file 2. Due to the similari es between the results from each of the three angles, we primarily discuss the 3D angle during feeding in this paper, but these addi onal measures serve to inform our analysis of direc onal tuning in terms of its horizontal and ver cal components.
For all subsequent analyses, we combined neurons in rM1 and cM1 as MIo and area 1/2, 3a/3b as SIo because of the small number of recorded neurons in some cor cal regions (Supplementary file 1, Table 1).We evaluated whether the distribu on of PDs across le to right direc ons differed between tasks and across cor cal regions.Intrinsic and extrinsic tongue muscles are involved in shaping the tongue (e.g., elonga on, broadening) and posi oning the tongue (e.g., protrusion/retrac on, eleva on/depression), respec vely.These muscles receive bilateral motor innerva on except for genioglossus.The straight tongue protrusion requires the balanced ac on of the right and le genioglossi while the lateral tongue protrusion requires the ac on of the contralateral genioglossus.Since genioglossus receive unilateral innerva on, we expected to see more neurons in the le MIo/SIo to have PDs for tongue movements that were le ward protrusion through the ac va on of the right genioglossus.Indeed, the PD distribu ons of direc onally tuned neurons during feeding had peaks towards tongue movements to the le , except for SIo of Monkey R (Fig. 6A).Similar results were found with the distribu ons of preferred yaw during feeding (Supplementary file 2, Fig. 4).While the PD distribu on in feeding were comparable between MIo and SIo in both subjects (circular k-test, p > 0.1), there was a significant difference during drinking between the PD distribu ons of MIo and SIo neurons in Monkey R (Chisquare, p < 0.001), but not in Monkey Y (p > 0.09).As in feeding, a majority of MIo and SIo neurons in Monkey Y had le protrusion as their PD.In contrast, propor on of MIo neurons with PDs for le and right protrusions were comparable in Monkey R, which might suggest involvement of muscles other than the right genioglossus during drinking.

Effects of nerve block
Sensa on plays a key role in tongue posi oning and movements for natural behaviors.During inges on, tac le feedback is necessary for loca ng the bolus, preven ng tongue bites, feeling where the drinking spout is, and iden fying when it is safe to swallow.To evaluate the role of oral sensa on, we used a bilateral oral nerve block to temporarily eliminate tac le sensa on in the oral cavity and observe how the control of tongue movement was impacted.Below we show how the loss of sensa on affected both tongue kinema cs and direc onal tuning of neurons during feeding and drinking.To verify that differences between the control and nerve block condi ons were due to the loss of sensory feedback and not as a result of other factors such as seda on and injec on, a sham experiment was conducted where saline was administered to the injec on sites instead of nerve block.No significant changes to tongue kinema cs were observed following the sham experiments (Supplementary file 1, Figs. 2 and 3).Tongue kinema cs.In feeding, the mean and overall spread of direc ons were significantly different between the control and nerve block condi ons (t-test, p < 0.01 and f-test, p < 0.001).There was a shi towards a smaller range of 3D direc ons in Monkey R, whereas there was a shi towards a broader distribu on in Monkey Y under the nerve block condi on (Fig. 7A).The posi ons of maximum protrusion of the tongue during drinking, i.e., the endpoints, were also affected by the loss of sensa on.These endpoints represent the planned target posi on of the tongue to receive the juice reward from a specific spout.In the control drinking task, the endpoints for each spout loca on were very dis nct.In contrast, the endpoints of tongue movements in nerve block exhibited a greater overlap across loca ons and more variance in all three axes of mo on, i.e., Posterior-Anterior, Inferior-Superior, and Le -Right (Fig. 7B).
Compared to the control, the trajectories of the tongue p in the nerve block condi on during drinking had a smaller range of Le -Right values.Visually the tongue trajectories toward the different spout loca ons were messier and less dis nct from each other as was observed in failed cycles where the tongue p missed the loca on of the correct spout (Fig. 8).Failed cycles are those whose endpoint posi ons are greater than 2 standard devia ons from the mean endpoint posi on.In both monkeys there was a significant increase in the average distance from the mean endpoint posi on, though this difference was much greater in Monkey R (Fig. 7C).We noted a

Figure 8. Effect of nerve block on drinking kinema cs in Monkey R. (A) Tongue p trajectories from star ng posi on to one of three drinking spouts in the control and nerve block condi ons. (B) Drinking trajectory endpoints, where the black dot represents the mean endpoint posi on.
difference between subjects in the frequency of failed cycles and the range of le -right tongue movements under nerve block.This may reflect a possible compensatory strategy of reaching the drinking spouts with an adjacent region of the tongue, instead of contac ng the right or le spout with the ipsilateral tongue in Monkey R.
Direc onal tuning of MIo and SIo neurons.Loss of oral sensa on also affected the propor on of direc onally tuned neurons and the overall distribu on of PDs, though the pa ern of changes differed between subjects.Following nerve block, MIo and SIo showed overall decreases in the propor on of direc onally modulated neurons in both tasks (Fig. 9A; Chi-square, MIo: p < 0.001, SIo: p < 0.01).We then verified whether these changes could be a ributed to neurons gaining or losing direc onal tuning in nerve block.The majority of neurons in MIo and SIo for both tasks remained direc onally tuned, and the propor ons of neurons that gained or lost direc onal tuning were similar between cor cal regions and subjects (Fig. 9B).

Figure 9. Effects of nerve block on direc onal tuning of OSMCx neurons during feeding and drinking tasks. (A) Percentage of direc onally tuned neurons in four areas: rM1 -rostral M1, cM1 -caudal M1, SC(1/2) -area 1/2, and SC(3a/3b) -area 3a/3b. Filled in bars represent control while empty bars represent nerve block. Error bars represent ±1 SE. (B) Percentage of MIo and SIo neurons which gained or lost direc onality with the addi on of nerve block.
The propor on of neurons that lost and gained direc onality were similar for MIo and SIo during feeding (Chi-square, p > 0.1).During drinking, more neurons lost direc onal tuning in SIo than in MIo in Monkey R (p < 0.01), but for Monkey Y the propor on of neurons that gained direc onal tuning was higher in SIo than MIo (p < 0.05).When comparing between behaviors, there were similar propor ons of neurons that gained and lost direc onal tuning for Monkey R (Chi-square, p > 0.1), but for Monkey Y there was a higher percentage of neurons that gained direc onality in both MIo and SIo during drinking than during feeding (p < 0.05).
As for PD distribu on, we found a significant shi in the mean PD in the nerve block condi on of the feeding task; the mean PD of MIo neurons in both subjects shi ed clockwise toward the center (0) and exhibited a more uniform distribu on (Fig. 10A; circular k-test, p < 0.01).The PD shi for the popula on of neurons in SIo was inconsistent, with only Monkey R showing a significant counterclockwise shi (p < 0.05).In drinking under nerve block, the PD distribu on in MIo shi ed from right to le in Monkey R and from le to right in Monkey Y (Fig. 10B; Chi-square, Y: p = 0.04), whereas the PD distribu on of SIo neurons shi ed towards the right in both animals (Chi-square, R: p = 0.02).

Popula on decoding of tongue direc on
Next, we evaluated the direc onal informa on contained in the popula on ac vity.We employed a k-nearest neighbor (KNN) to predict the tongue direc on from the firing rates of each neuron over a 100 ms interval.The KNN classifier was able to decode the direc on of tongue movements above chance level for both feeding and drinking behaviors.Due to the large variability in the population size of MIo and SIo neurons which could affect the results, we compared decoding accuracies by downsampling the number of neurons used for decoding to that of the smallest recorded population, N = 28 (Fig. 11).Through mul ple linear regression, the effects of the behavior (feeding/drinking), region (MIo/SIo), condi on (control/nerve block), and interac ons between these factors on decoding accuracy were evaluated.The performance of the decoder was on average 20% higher in the drinking task than in the feeding task.Decoding accuracy was greater using MIo than SIo by 13%, and in the absence of tac le sensa on there was no significant decrease in the performance of the KNN classifier.Between subjects, decoding accuracy was similar in the feeding task but 17% higher for Monkey R than Monkey Y in the drinking task, indicating a higher inter-subject variability in directional information for the drinking task.

Discussion
The aim of this study was to determine how 3D direc onal informa on is represented by MIo and SIo neurons during natural feeding and drinking behaviors, and how it is affected by the loss of tac le sensa on.We found high propor ons of direc onally tuned neurons and above chance level decoding of direc ons from neuronal ac vity.When comparing control sessions to sessions preceded by the bilateral nerve block, we found significant changes in the direc on of tongue movement, the propor on of direc onally tuned neurons, and the distribu on of the PDs of OSMCx neurons.

Direc onal tuning in the oral sensorimotor cortex
This is the first study to inves gate direc onal tuning of OSMCx neurons to 3D tongue direc on con nuously over me during macaque feeding and drinking.Unlike previous similar studies, the monkeys were not trained to reach specific targets and were instead allowed to eat and drink rela vely naturally.In prior work, it was found that the OSMCx encodes direc on similarly to the arm region during a trained tongue protrusion task (Murray and Sessle, 1992;Sessle et al., 2005a;Arce et al., 2013).In the present study, results from the more natural drinking were consistent with findings in the tongue protrusion task.We found similarly high propor ons of direc onally modulated neurons, and the PD distribu ons in the control condi on were similar to those of the trained tongue protrusion.More importantly, by comparing the two natural behaviors, we found that the direc onal informa on in OSMcx was lower in feeding than in our drinking task, indica ng a higher cor cal engagement in behaviors characterized by a greater degree of voli on.
Interes ngly, the propor on of direc onally tuned neurons in the feeding task exhibited a significant disparity between MIo and SIo, sugges ng a higher direc onal informa on in MIo for tongue movements in feeding.The difference between the direc onal tuning of neurons in feeding compared to drinking suggests that direc onal tuning proper es of neurons is modifiable by the behavioral task.In the drinking task, the tongue moves to discrete loca ons to receive the juice reward, thus there is greater involvement of SIo in determining tongue direc on and reliance on sensory feedback.In contrast, tongue movements during feeding may have minimal inten on or voli on in moving the tongue in a specific direc on.We considered that the difference in the direc onal tuning of SIo neurons between the two behaviors could be due to the different me intervals used for each task since the period around minimum tongue protrusion in the drinking may contain more of the sensory inputs from the previous lick.However, when sampling spiking ac vity from an earlier me period in feeding, the percentage of direc onally tuned SIo neurons was s ll significantly lower than MIo (Chi-square, p < 0.001, data not shown).Shi ing the me period of the drinking trials to be a er minimum protrusion also did not lead to a significant difference between MIo and SIo (p > 0.1).Our results suggest that the somatosensory cortex may be less involved than the motor areas during feeding, possibly because it is a more ingrained and stereotyped behavior as opposed to tongue protrusion or drinking tasks, where the subject must specifically reach out with the tongue to feel the spout and juice reward.It has been found that propriocep ve feedback is suppressed by spinal interneurons during locomo on, another semiautoma c behavior, to promote smooth and rhythmic movements (Koch et al., 2017;Dallmann et al., 2023).A similar mechanism may exist for feeding, where unnecessary or confounding sensory informa on about tongue movement is more inhibited during chewing or swallowing.
We addi onally found different distribu ons of PDs.This signifies that there are more neurons in the le hemisphere contribu ng toward one direc on of tongue movement, sugges ng that there is some laterality in the PDs of OSMCx neurons that varies between individuals.The preferred chew side was the same for both monkeys (i.e., right side), so this finding may be somewhat contrary to previous results in humans examined using fMRI showing that hemispheric differences in sensorimotor ac vity during voluntary tongue movements are related to the preferred chew side (Shinagawa et al., 2003).It is possible that the difference between the two subjects is related to the difference in recording loca ons, with Monkey Y's being more lateral and therefore closer to the swallow area of the cortex than Monkey R's (Supplementary file 1, Fig. 1).Monkey Y had a higher propor on of neurons that were tuned to tongue direc on during feeding compared to Monkey R (Supplementary file 1, Fig. 4), but fewer during drinking.An avenue for further study could be a unilateral nerve block on the preferred side to measure how the unaffected side of the tongue compensates for the lack of sensa on in the affected side.A previous study found that unilateral lingual nerve transec on in pigs alters the coordina on of the ipsilateral tongue side during chewing (Montuelle et al., 2020).The tongue is a complex group of muscles, with intrinsic muscles primarily contribu ng to the shape and size of the tongue and extrinsic muscles contribu ng more to the posi oning of the tongue.Therefore, it is possible that the neurons which are strongly tuned to tongue direc on have direct connec ons to the extrinsic muscles on the ipsilateral side.Looking at how each side of the tongue responds independently to unilateral nerve block, and how this interacts with direc onal preference may give us more informa on about how the unique structure of the tongue is coordinated.

Effects of the loss of tac le feedback
It was previously found that the administra on of bilateral nerve block impaired feeding performance and tongue jaw coordina on, and that it slowed the feeding sequence (Laurence-Chasen et al., 2022).The present study shows that direc onal movement of the tongue (kinema cs) and the spiking ac vity of MIo and SIo were also affected.In the absence of sensory feedback, there were decreased propor ons of direc onally tuned neurons, shi s in the distribu ons of PDs, less dis nct groups of drinking endpoints, more failed drinking cycles, and shi s in the distribu on of tongue direc ons across feeding cycles.The reduc on in direc onally tuned neurons in feeding may be a ributed to a more cau ous approach to avoid injury in the absence of sensory feedback.This may be indica ve of a reversion to previously known feeding pa erns, causing the monkey to be less likely to adjust the feeding sequence when unable to feel the loca on of the bolus.
Our results demonstrate the importance of tac le feedback in direc ng tongue movement especially during drinking.Tac le feedback allows for adjustments of the direc on of tongue movement to achieve contact with the spout within one or two licks.Without this informa on, the animal is only guided by whether the juice reward was received or not.In this case the subjects may u lize gustatory feedback to aid in loca ng the correct spout.In a recent optogene c inhibi on study on licking in mice, it was found that the tongue/jaw regions of the somatosensory cortex were necessary for proper tongue targe ng but not for the core motor capabili es of the tongue (Xu et al., 2022).The decrease in the range of tongue mo on that we observed is therefore most likely due solely to the loss of sensory feedback, not a loss of motor func on itself.It is also worth no ng that our drinking setup does not completely eliminate visual feedback for the monkey, which could also contribute to readjus ng tongue posi on.However, oral sensory loss alone had a significant effect on the monkeys' performance in this task, and tongue movements are not typically guided by visual inputs as the tongue is usually not visible within the oral cavity.
There were varying effects of the nerve block on direc onal tuning.The propor on of direc onally tuned MIo neurons increased in Monkey R but decreased in Monkey Y during feeding, while the propor on of direc onally tuned SIo neurons decreased in Monkey R but increased in Monkey Y during drinking.An increased propor on of neurons were tuned to pitch in Monkey Y with nerve block, whereas there was a decrease in Monkey R.There was no significant shi in the distribu on of PDs in only the SIo of Monkey Y during both feeding and drinking with nerve block.Monkey Y also did not exhibit the same increase in failed cycles that was observed in Monkey R and did not display as significant a shi in the propor on of neurons that were direc onally tuned during drinking as Monkey R did.Interes ngly, there was a rela vely large propor on (40%) of SIo neurons in Monkey Y that gained direc onal tuning following sensory loss compared to Monkey R (8%) during drinking.The loss of sensa on affected these proper es to a lesser extent in Monkey Y, possibly due to differences in the array loca ons or differing compensatory strategies between the two subjects.Due to the high variability between individuals in their response to nerve block, studying addi onal subjects would expand our understanding of how the OSMCx adapts to coordinate tongue movement following sensory disrup on.
A limita on of the present study is that head posi on and hand movement were restrained, especially since there may be significant interac on between hand and orofacial regions while handling food or drink.The hand and orofacial areas are next to each other in the cortex and highly interconnected (Forrester and Rodriguez, 2015), and researchers have found an area in mice that coordinates hand-to-mouth movements during natural feeding (An et al., 2022).Fully natural feeding would involve holding food up to the mouth, as well as free head movement, which would make tracking of the marker posi ons difficult under this experimental setup.Further improvements in our ability to track tongue movements would be necessary to study more complex feeding sequences.

Popula on decoding of tongue direc on
The ability of a popula on of neurons to decode tongue direc on from spiking ac vity over a short me interval reveals the extent to which the OSMCx is informed of tongue direc on during natural behaviors, and thus, it has implica ons for the control of neuroprostheses.The direc on of voluntary tongue protrusion was able to be decoded from simultaneously recorded MIo and SIo popula ons (Arce et al., 2013).Here, we showed that this could be applied to a cyclic, naturalis c behavior, and that instantaneous 3D tongue direc on could be decoded.We observed an increase of up to 10% in decoder performance with the full popula ons of recorded neurons compared to the results from only 28 neurons, so we expect that decoding accuracy would increase to a similar extent with a much larger popula on (>100 neurons).Although downsampling leads to an overall decrease in decoding accuracy, it allows for the comparison across other variables.The higher decoding accuracy in drinking than in feeding suggests that direc onal informa on in OSMCx neurons is higher in drinking from varying spout loca on.As expected, decoding from MIo yielded higher accuracy than SIo in both behaviors, though the dis nc on was greater during feeding, consistent with our direc onal tuning results.These results support the well-established role of MIo in the control of movement parameters, especially direc on.
The changes to decoding accuracy with nerve block were less significant than the other changes we observed.There was a slight decrease in decoding accuracy with nerve block in MIo and no difference overall in SIo, indica ng that MIo and SIo s ll contain substan al informa on about tongue direc on even without tac le feedback.While it was expected that MIo may s ll be informed about the movement of the tongue, it is surprising that performance using SIo remained consistent and even increased with nerve block.This may be evidence that another area is supplying SIo with informa on to compensate for the lack of tac le feedback, such as informa on from the taste buds.

Clinical implica ons
This study offers new informa on about the important role of sensorimotor integra on in controlling tongue direc on during natural behaviors.There is a high degree of direc onal informa on contained in the spiking ac vity of the orofacial cortex, especially in the motor areas.The effect of the bilateral nerve block serves to enhance our understanding of the processes affected by oral sensorimotor dysfunc ons such as trigeminal neuropathies.It demonstrates the importance of oral sensa on for suppor ng the full range of direc onal mo on, but also shows that significant direc onal informa on can be extracted even in the absence of tac le feedback.This type of knowledge can inform the diagnosis and rehabilita on of orolingual dysfunc ons.There have also been advancements in brain-computer interface (BCI) by decoding the real-me signals of arm region of the motor cortex to control prosthe c arm movement (Collinger et al., 2013;Vilela and Hochberg, 2020) or muscle s mula on (Ethier and Miller, 2015), as well as efforts to restore sensory feedback by s mula ng correct areas of somatosensory cortex in response to sensors on a prosthe c (Tabot et al., 2013;Flesher et al., 2021).Therefore, informa on about how a different feature of movement is encoded in the OSMCx may prove helpful in the development of therapies or so prosthe cs for people with impaired orolingual func on or glossectomy.

Figure 1 .
Figure 1.Direc on of tongue mo on in each behavioral task.(A) Schema c of the loca on of three spouts, le (L), middle (M), and right (R), for the drinking task.Tongue direc on was categorized based on spout loca on.(B) Calcula on of 3D tongue direc on during feeding.θ is the instantaneous 3D direc on of the tongue p over a 100 ms interval between its posi ons at t1 and t2, where t1 = 0 and t2 = t1 + 100.The do ed line shows the actual trajectory during this interval.

Figure 4 .
Figure 4. Cosine tuning of MIo and SIo neurons.(A) Distribu on of 3D preferred direc ons in unit sphere for neurons that fit the tuning func on during feeding, combined for both subjects.The origin represents the start of a movement.Color bar represents posterior-anterior axis.(B) Distribu on of the index for the depth of direc onal tuning, combined for both subjects.

Figure 5 .
Figure 5. Direc onal tuning to yaw and pitch during feeding.(A) Firing rate maps of a neuron in MIo and in SIo across yaw and pitch angles.Firing rates were averaged across all 100 ms feeding intervals within a 10° range.(B) Proportion of neurons tuned to yaw and pitch, combined for both subjects.Recordings were taken from four areas of the OSMCx: rM1 -rostral M1, cM1 -caudal M1, SC(1/2) -area 1/2, and SC(3a/3b) -area 3a/3b.Error bars represent ±1 SE.

Figure 6 .
Figure 6.Distribu on of PDs in MIo (yellow) and SIo (purple) neurons during control feeding (A) and drinking (B).For the feeding task, polar plots are split into 10 bins with thick colored lines represen ng the mean PD.For the drinking task, error bars represent ±1 SE.

Figure 7 .
Figure 7. Effect of nerve block on direc on of tongue movement.(A) Distribu on of tongue direc ons during feeding.(B) Variance in 3D trajectory endpoints during drinking (Posterior-Anterior, Inferior-Superior, Le -Right) for each direc on: le (L), middle (M), right (R). (C) Varia on in the distance of drinking endpoint posi ons from the mean endpoint.Le halves of hemi-violins (black) are control and right halves (red) are nerve block for an individual.Horizontal black lines represent the mean and horizontal red lines the median.Results of two-tailed t-test and f-test are indicated by asterisks and crosses, respec vely: *, † p < 0.05; **, † † p < 0.01; ***, † † † p < 0.001.

Figure 10 .
Figure 10.Effects of nerve block on the distribu on of PDs of MIo (yellow) and SIo (purple) neurons.(A) For the feeding task, polar plots are split into 10° bins with thick colored lines represen ng the mean PD.Significant circular concentra on test (k-test) comparing control and nerve block are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.(B) For the drinking task, error bars represent ±1 SE.Filled in bars represent control while empty bars represent nerve block.

Figure 11 .
Figure 11.Decoding accuracies from neuronal populations of equal size (N=28).Data shown separately for each subject, behavioral task, and condition.The dashed line signifies equal decoding performance for MIo and SIo.Chance level is 33.33%.Decoding accuracies from full populations are included in Supplementary file 1, Fig. 5.